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J. Biol. Chem., Vol. 278, Issue 3, 1647-1655, January 17, 2003

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Construction of a Deep-rough Mutant of Burkholderia cepacia ATCC 25416 and Characterization of Its Chemical and Biological Properties^*

Sabine Gronow^§, Christian Noah, Antje Blumenthal^¶, Buko Lindner, and Helmut Brade

From the Divisions of  Medical and Biochemical Microbiology, ^¶ Molecular Infection Biology, and  Biophysics, Research Center Borstel, Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany

Burkholderia cepacia is a bacterium with increasing importance as a pathogen in patients with cystic fibrosis. The deep-rough mutant Ko2b was generated from B. cepacia type strain ATCC 25416 by insertion of a kanamycin resistance cassette into the gene waaC encoding heptosyltransferase I. Mass spectrometric analysis of the de-O-acylated lipopolysaccharide (LPS) of the mutant showed that it consisted of a bisphosphorylated glucosamine backbone with two 3-hydroxyhexadecanoic acids in amide-linkage, 4-amino-4-deoxyarabinose (Ara4N) residues on both phosphates, and a core oligosaccharide of the sequence Ara4N-(1  8) D-glycero-D-talo-oct-2-ulosonic acid (Ko)-(2  4)3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The mutant allowed investigations on the biosynthesis of the LPS as well as on its role in human infection. Mutant Ko2b showed no difference in its ability to invade human macrophages as compared with the wild type. Furthermore, isolated LPS of both strains induced the production of tumor necrosis factor  from macrophages to the same extent. Thus, the truncation of the LPS did not decrease the biological activity of the mutant or its LPS in these aspects.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ302064 and AJ420777.

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